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嗜水气单胞菌菌落多重PCR方法的建立及应用
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安徽省农业科学院科研团队计划项目(18C0513);安徽省重点研究和开发计划项目(201904f06020021)


Establishment and application of colony multiplex PCR for Aeromonas hydrophila
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    摘要:

    【背景】嗜水气单胞菌(Aeromonas hydrophila)对水产动物、畜禽和人类均有致病性。基因表达的溶血素、气溶素和肠毒素是重要毒力因子,在致病性嗜水气单胞菌早期检测及防治中尤为重要。目前采用菌落直接提取DNA用于多重PCR研究的相关报道较少。【目的】基于菌落PCR方法建立针对嗜水气单胞菌溶血性基因、肠毒素基因和16s rRNA基因特异性片段(5个基因片段)的多重PCR快速检测方法。【方法】采用选择性RS (Rimler-Shotts)培养基对样品中嗜水气单胞菌有效富集分离和辨认,建立并优化嗜水气单胞菌16s rRNA、astaltaerAact这5个基因的多重PCR方法,比较菌落PCR中DNA模板不同提取方法对多重PCR扩增结果的影响,并检测该方法对维氏气单胞菌、温和气单胞菌、杀鲑气单胞菌的特异性。【结果】通过对RS培养基上单菌落的16s rRNA基因鉴定,初步判定嗜水气单胞菌和其他可培养菌的菌落形态,对其富集程度进行可视化辨别。多重PCR反应体系优化结果显示,引物浓度最优配比为16s rRNA:ast:alt:aerA:act=1:2:2:3:4。菌落PCR结果显示,新鲜菌液采用煮沸冷冻离心法和煮沸离心法均能扩增出清晰条带,采用单菌落处理则需煮沸冷冻离心法。该多重PCR方法具有特异性。【结论】利用菌落多重PCR可以不通过试剂盒抽提获得DNA模板,简便、直观地检测嗜水气单胞菌及其毒力基因,具有特异性。

    Abstract:

    [Background] Aeromonas hydrophila is pathogenic to aquatic animals, livestock, poultry, and human. Hemolysin, aerolysin, and enterotoxin are major virulence factors particularly important in the early detection and prevention of A. hydrophila. There are few reports using colony PCR to extract templates for multiplex PCR. [Objective] Based on colony PCR, a multiplex PCR assay was established for rapid detection of five genes including hemolysin gene, enterotoxin gene, and 16s rRNA gene of A. hydrophila. [Methods] We used the selective Rimler-Shotts (RS) medium to enrich, isolate, and identify A. hydrophila in the sample. Subsequently, we established and optimized the multiplex PCR conditions for the 16S rRNA, ast, alt, aerA, and act of A. hydrophila and compared the results of multiplex PCR with the DNA templates extracted by different methods in colony PCR. Finally, we evaluated the specificity of the established method by using A. veronii, A. sobria and A. salmonicida. [Results] After 16S rRNA identification of single colonies on RS plates, the colony morphology of A. hydrophila and other cultivable bacteria was preliminarily characterized, and the enrichment degree of A. hydrophila was visually identified. The optimization results of the multiplex PCR system showed that the optimal primer concentration ratio was 16s rRNA:ast:alt:aerA:act=1:2:2:3:4. PCR for fresh bacterial liquids treated with both boiling-refrigerated centrifugation method and boiling-centrifugation method could produce clear bands, while the PCR of single colony required the former method. The established multiplex PCR method was specific. [Conclusion] Colony multiplex PCR can be used to easily and intuitively detect A. hydrophila and its virulence genes without the need of kit extraction of DNA templates, and it was specific.

    参考文献
    [1] 陆承平. 致病性嗜水气单胞菌及其所致鱼病综述[J]. 水产学报, 1992, 16(3): 282-288 Lu CP. Pathogenic Aeromonas hydrophila and the fish diseases caused by it[J]. Journal of Fisheries of China, 1992, 16(3): 282-288 (in Chinese)
    [2] 杨守明, 王民生. 嗜水气单胞菌及其对人的致病 性[J]. 疾病控制杂志, 2006, 10(5): 511-514 Yang SM, Wang MS. Aeromonas hydrophila and its pathogensis to humans[J]. Chinese Journal of Disease Control & Prevention, 2006, 10(5): 511-514 (in Chinese)
    [3] 沈锦玉. 嗜水气单胞菌的研究进展[J]. 浙江海洋学院学报(自然科学版), 2008, 27(1): 78-86 Shen JY. Research progresss on Aeromonas hydrophila[J]. Journal of Zhejiang Ocean University: Natural Science, 2008, 27(1): 78-86 (in Chinese)
    [4] Pridgeon JW, Klesius PH. Molecular identification and virulence of three Aeromonas hydrophila isolates cultured from infected channel catfish during a disease outbreak in west Alabama (USA) in 2009[J]. Diseases of Aquatic Organisms, 2011, 94(3): 249-253
    [5] Hossain MJ, Sun DW, McGarey DJ, Wrenn S, Alexander LM, Martino ME, Xing Y, Terhune JS, Liles MR. An Asian origin of virulent Aeromonas hydrophila responsible for disease epidemics in United States-farmed catfish[J]. mBio, 2014, 5(3): 1-7
    [6] 符贵红, 肖丹, 胡鲲, 杨先乐. 鲫源嗜水气单胞菌毒力基因多重PCR检测及ERIC-PCR分子分型[J]. 海洋渔业, 2014, 36(6): 549-556 Fu GH, Xiao D, Hu K, Yang XL. Multiplex PCR and ERIC-PCR genotype in virulence genes of Aeromonas hydrophila in Crucian carp[J]. Marine Fisheries, 2014, 36(6): 549-556 (in Chinese)
    [7] 余文杰. 多重PCR技术检测食用鱼类产品中β-溶血性嗜水气单胞菌毒性基因研究[J]. 现代农业科技, 2019(11): 217-218, 225 Yu WJ. Multiplex PCR detection of haemolysin genes in β-haemolytic Aeromonas hydrophila strains isolated from edible fish product[J]. Modern Agricultural Science and Technology, 2019(11): 217-218, 225 (in Chinese)
    [8] 韦信贤, 杨先乐, 童桂香, 吴祥庆, 谢宗升, 黄国秋, 廖永志, 叶欣宇, 黎小正. 四重PCR检测致病性嗜水气单胞菌[J]. 中国人兽共患病学报, 2013, 29(6): 587-593 Wei XX, Yang XL, Tong GX, Wu XQ, Xie ZS, Huang GQ, Liao YZ, Ye XY, Li XZ. Detection of pathogenic Aeromonas hydrophila by quadruple PCR[J]. Chinese Journal of Zoonoses, 2013, 29(6): 587-593 (in Chinese)
    [9] 凌空, 丁诗华, 金娟, 吴兴镇. 一种检测大鲵致病性嗜水气单胞菌的四重PCR法[J]. 生物技术通报, 2014(9): 201-207 Ling K, Ding SH, Jin J, Wu XZ. Detection of pathogenic Aeromonas hydrophila in Andrias davidianus by quadruple PCR[J]. Biotechnology Bulletin, 2014(9): 201-207 (in Chinese)
    [10] 熊静, 赖晓健, 余钦, 郭松林, 徐继松, 黄文树. 7株鳗鲡致病性气单胞菌毒力基因胞外产物及其活性比较[J]. 华中农业大学学报, 2017, 36(1): 76-85 Xiong J, Lai XJ, Yu Q, Guo SL, Xu JS, Huang WS. Analysis of virulence genes, extracellular products and activities among seven Aeromonas strains isolated from eels[J]. Journal of Huazhong Agricultural University, 2017, 36(1): 76-85 (in Chinese)
    [11] 胡萌. 江苏地区气单胞菌分离鉴定及强毒株生物学特性分析[D]. 南京: 南京农业大学硕士学位论文, 2012 Hu M. Isolation and identification of Aeromonas from Jiangsu and characterizations of virulent strains[D]. Nanjing: Master’s Thesis of Nanjing Agricultural University, 2012 (in Chinese)
    [12] 范腾飞. 气单胞菌的毒力基因检测及环境因子对毒力基因表达的影响[D]. 合肥: 合肥工业大学硕士学位论文, 2013 Fan TF. Detection of virulence genes in Aeromonas strains and the influence of environmental factors on virulence genes expression[D]. Hefei: Master’s Thesis of Hefei University of Technology, 2013 (in Chinese)
    [13] 付乔芳, 邱军强, 胡鲲, 杨先乐, 安健. 嗜水气单胞菌国内分离株的毒力因子分布与致病性相关性分 析[J]. 生物学杂志, 2011, 28(6): 53-57 Fu QF, Qiu JQ, Hu K, Yang XL, An J. The analyse of virulence factors-pathogenicity relationships of Aeromonas hydrophila strains isolated from China[J]. Journal of Biology, 2011, 28(6): 53-57 (in Chinese)
    [14] 王一娟. 江苏地区大宗淡水鱼养殖池塘气单胞菌分离、鉴定和遗传多样性研究[D]. 南京: 南京农业大学硕士学位论文, 2010 Wang YJ. Studies on isolation, Identification&Genetic diversity of Aeromonas from conventional freshwater fish ponds in Jiangsu Province[D]. Nanjing: Master’s Thesis of Nanjing Agricultural University, 2010 (in Chinese)
    [15] Wong CYF, Heuzenroeder MW, Flower RLP. Inactivation of two haemolytic toxin genes in Aeromonas hydrophila attenuates virulence in a suckling mouse model[J]. Microbiology: Reading, England, 1998, 144(Pt2): 291-298
    [16] 朱大玲. 嗜水气单胞菌毒力基因及基因工程疫苗[D]. 武汉: 中国科学院研究生院(水生生物研究所)博士学位论文, 2006 Zhu DL. The virulence genes and genetic engineering vaccines of Aeromonas hydrophila[D]. Wuhan: Doctoral Dissertation of Chinese Academy of Sciences (Institute of Hydrobiology), 2006 (in Chinese)
    [17] Cascón A, Fregeneda J, Aller M, Yugueros J, Temprano A, Hernanz C, Sánchez M, Rodríguez-Aparicio L, Naharro G. Cloning, characterization, and insertional inactivation of a major extracellular serine protease gene with elastolytic activity from Aeromonas hydrophila[J]. Journal of Fish Diseases, 2000, 23(1): 49-59
    [18] 储卫华, 陆承平. PCR扩增特异性16SrDNA和溶血素基因检测致病性嗜水气单胞菌[J]. 水产学报, 2005, 29(1): 79-82 Chu WH, Lu CP. PCR detection of pathogenic Aeromonas hydrophila by specific 16S rDNA and aerolysin gene[J]. Journal of Fisheries of China, 2005, 29(1): 79-82 (in Chinese)
    [19] 高建忠, 魏雪, 童琰, 黄玉邦. MGB探针实时定量PCR检测致病性嗜水气单胞菌[J]. 安徽农业科学, 2009, 37(19): 8911-8913 Gao JZ, Wei X, Tong Y, Huang YB. Application of real-time PCR for detection of Aeromonas hydrophila by using MGB fluorescence probe[J]. Journal of Anhui Agricultural Sciences, 2009, 37(19): 8911-8913 (in Chinese)
    [20] Jenkins JA, Taylor PW. An alternative bacteriological medium for the isolation of Aeromonas spp.[J]. Journal of Wildlife Diseases, 1995, 31(2): 272-275
    [21] Aboyadak IM, Ali NG, Goda AM, Saad W, Salam AM. Non-selectivity of R-S media for Aeromonas hydrophila and TCBS media for Vibrio species isolated from diseased Oreochromis niloticus[J]. Journal of Aquaculture Research & Development, 2017, 8(7): 1-5
    [22] 庞茂达. 嗜水气单胞菌流行株基因组特征及毒力相关基因研究[D]. 南京: 南京农业大学博士学位论文, 2015 Pang MD. Genomic characteristics and virulence- associated genes analysis of Aeromonas hydrophila[D]. Nanjing: Doctoral Dissertation of Nanjing Agricultural University, 2015 (in Chinese)
    [23] Tomás JM. The main Aeromonas pathogenic factors[J]. ISRN Microbiology, 2012, 2012: 256261
    [24] Cascón A, Anguita J, Hernanz C, Sánchez M, Fernández M, Naharro G. Identification of Aeromonas hydrophila hybridization group 1 by PCR assays[J]. Applied and Environmental Microbiology, 1996, 62(4): 1167-1170
    [25] Chopra AK, Houston CW. Enterotoxins in Aeromonas- associated gastroenteritis[J]. Microbes and Infection, 1999, 1(13): 1129-1137
    [26] 陈书霞, 王晓武, 房玉林. 单菌落PCR法直接快速鉴定重组克隆[J]. 微生物学通报, 2006, 33(3): 52-56 Chen SX, Wang XW, Fang YL. Rapid characterization of recombination clone by PCR screening of individual bacterial colonies[J]. Microbiology China, 2006, 33(3): 52-56 (in Chinese)
    [27] 徐丽, 蔡俊鹏. 菌落PCR方法的建立及其与常规PCR方法的比较[J]. 华南理工大学学报: 自然科学版, 2004, 32(5): 51-55 Xu L, Cai JP. Establishment of colony PCR method and its comparison with conventional PCR method[J]. Journal of South China University of Technology: Natural Science, 2004, 32(5): 51-55 (in Chinese)
    [28] 潘渠, 杨维华, 王颖, 余小平, 陈恬, 陈玮. 设计乳杆菌特异性引物并运用菌落PCR技术快速检出和鉴定四川泡菜中的乳杆菌[J]. 微生物学通报, 2011, 38(8): 1300-1305 Pan Q, Yang WH, Wang Y, Yu XP, Chen T, Chen W. Rapid detection and identification Lactobacillus from Sichuan pickle by colony PCR using Lactobacillus-specific primers[J]. Microbiology China, 2011, 38(8): 1300-1305 (in Chinese)
    [29] 李伟杰, 于建慧, 魏财文, 蒋桃珍. 产气荚膜梭菌毒素型菌落多重PCR方法的建立及应用[J]. 西北农业学报, 2016, 25(6): 823-827 Li WJ, Yu JH, Wei CW, Jiang TZ. Establishment and application of colony multiplex PCR assay for toxinotyping Clostridium perfringens[J]. Acta Agriculturae Boreali-Occidentalis Sinica, 2016, 25(6): 823-827 (in Chinese)
    [30] 董洁, 杨晓静, 苗承霞, 沈晶, 杨柳, 姜艳芬, 许信刚. 产气荚膜梭菌菌落多重PCR方法的建立及初步应用[J]. 中国兽医学报, 2013, 33(12): 1842-1847 Dong J, Yang XJ, Miao CX, Shen J, Yang L, Jiang YF, Xu XG. Development and preliminary application of colony multiplex PCR for serotyping of Clostridium perfringens strains[J]. Chinese Journal of Veterinary Science, 2013, 33(12): 1842-1847 (in Chinese)
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张静,王永杰,陈红莲,鲍俊杰,孙雯. 嗜水气单胞菌菌落多重PCR方法的建立及应用[J]. 微生物学通报, 2022, 49(2): 841-850

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  • 收稿日期:2021-07-16
  • 最后修改日期:2021-08-08
  • 在线发布日期: 2022-02-21
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